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Abstract
As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all α-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.
| Abbreviations | ||
| FSC | = | forward scatter |
| SSC | = | side scatter |
| C.R. | = | Congo red |
| AFM | = | atomic force microscopy |
| TEM | = | transmission electron microscopy |
| GA | = | glutaraldehyde |
| TFE | = | 2,2,2-trifluoroethanol |
| EtOH | = | ethanol |
| HFIP | = | 1,1,1,3,3,3-hexafluoroisopropanol |
| CM-papain | = | carboxy-methylated papain |
| PBS | = | phosphate-buffered saline |
| FPLC | = | fast protein liquid chromatography |
| Abbreviations | ||
| FSC | = | forward scatter |
| SSC | = | side scatter |
| C.R. | = | Congo red |
| AFM | = | atomic force microscopy |
| TEM | = | transmission electron microscopy |
| GA | = | glutaraldehyde |
| TFE | = | 2,2,2-trifluoroethanol |
| EtOH | = | ethanol |
| HFIP | = | 1,1,1,3,3,3-hexafluoroisopropanol |
| CM-papain | = | carboxy-methylated papain |
| PBS | = | phosphate-buffered saline |
| FPLC | = | fast protein liquid chromatography |